Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:27001516+27001627 112bp GAGAATTGAGTTGGTACCGAGGA GTCTGGCTTCTGTCACAACCT
Mut= 120 bp
Wt= 112 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TTCTGTTCTGTATCTATAAAAGGTCTCCAACAGGGTCGGAAGGGAATAGAGTGCCTGCCCTAGGTGGTTCACTCCCAGTGAGAAATAGTATTTGTTTCAGAAGACCCTCTGTGTACATGTGTCACGTCAGGCATTCTAAATCTCTCCTcactgaaaaatgaatggtagagttcaaaatgtatgttctctatcaccaaggcttctctctctctctctctctctctctccctctctctcccttcccccatcctctttttttttgggggggggggaatgggggagacaattcttttgtgaaaccttgaagaattccctaggaaaattcaaagcaagttactgaaatgacttttttccagaactttgtggaacataggcaatataagatggaccttcttcttctgattttagagaaaggtggcaacccacccctacatgccccagatcttcccagtgagacttcatttccttgtccagtggatcatccaaggccagaagaaagggctccgaaatatgaaaattatttttcagcctccgaaacacgctctttgaaaagagaattgagttggtaccgaggacgaaggccaagaaccgctttcacccagaaccaggtgagaagatttttcagccaatagtgacaaCACAAAGGTTGTGACAGAAGCCAGACACTCTGGGGTCCCAGTGTAGAAGTGGCATTAGTGAAAGGGTGGACTCATAGTTTAACAGTCTCTGGGCAACCAATTGTTTTAAAATGCTTTTGGATTAAAGGCCCAAATTTAAGTGCATTTTTCATTTGAAAGCCCAATT
This mutation is a 490 bp deletion beginning at Chromosome 14 position 27,001,112 bp and ending after 27,001,601 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45083 | GAG AAT TGA GTT GGT ACC GAG GA | Wild type Forward | A | |||
45084 | GTC TGG CTT CTG TCA CAA CCT | Common | A | |||
45085 | CTG CCC TAG GTG GTT CAC TC | Mutant Forward | A | |||
45086 | Fluorophore-1 | AGG CCA AGA ACC GCT TTC | Quencher-1 | WT Probe | ||
45087 | Fluorophore-2 | CAT GTG TCA CGT CAG GCA TT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45083 | 0.40 uM |
45084 | 0.40 uM |
45085 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |